The determination of nucleotide sequences in DNA material is fundamental to the field of genetic research and investigation, and to the synthesis of DNA materials. Methods currently used for this purpose, however, have technical limitations. For example, the well-established and widely practised Sanger dideoxy method for DNA sequencing relies upon generating randomly-terminated fragmental complements of the target DNA and electrophoretic separation of the fragments for analysis. Because of the limits on the sensitivity of the electrophoretic separation, the Sanger method is effectively limited in practice to sequencing of DNA samples having a maximum of about 500 nucleotide bases. Many genes of interest are longer than 500 bases in sequence.
Reducing the length of target DNA sequences inserted into M13 or plasmid vectors has become a routine step for deoxynucleotide sequence analysis. The commonly used methods involve deletion with exonucleases or restriction enzymes. Most of these methods require purification of double stranded or single stranded DNA, and sufficient knowledge of the restriction map of the target DNA to allow selection and use of a unique restriction site that is absent inside the target DNA.
It is an object of the present invention to provide a novel process for preparing deletion mutants of target DNA, useful in sequencing the DNA.